FSHbeta mRNA Research Articles

Corticosterone selectively increases follicle-stimulating hormone beta-subunit messenger ribonucleic acid in primary anterior pituitary cell culture without affecting its half-life.

We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro. In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA). The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript. Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin. Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h. At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis. Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA. To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h. The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs. In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells. The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period. Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability. We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization.

The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph.

Steroidogenic factor-1 (SF-1), also known as adrenal-4-binding protein, is a transcription factor that is important for the differentiation of steroidogenic tissues. We investigated whether SF-1 is expressed in specific hormone-producing cell types in the human pituitary and its adenomas. Pituitary adenomas (n = 35) were collected at the time of surgery, and normal adenohypophyses were obtained from autopsies; 3 corticotroph adenomas were excluded because pit-1 messenger ribonucleic acid (mRNA) expression indicated contamination by nontumorous elements. Expression of SF-1 mRNA was determined by reverse transcription-PCR. SF-1 protein was localized with immunocytochemistry. By reverse transcription-PCR, SF-1 mRNA was found in the nontumorous pituitary and in pituitary adenomas expressing gonadotropins. All 8 gonadotroph adenomas had a strong signal for SF-1. SF-1 mRNA was also detected in 2 of 3 corticotroph adenomas, 2 of 13 somatotroph/mammosomatotroph adenomas, 1 of 6 lactotroph adenomas, and 2 silent subtype 3 adenomas; however, in most of the positive tumors there was also positivity for FSH beta and/or LH beta mRNA, suggesting that contaminating nontumorous gonadotrophs may be the source of SF-1 mRNA signal. SF-1 protein was localized by immunocytochemistry in the nuclei of scattered cells of the nontumorous adenohypophysis that were shown to be gonadotrophs with double immunostaining and in gonadotroph adenomas; nuclear staining was not found in other adenoma types, except in areas shown to contain trapped nontumorous tissue. We conclude that SF-1 expression correlates with the expression of gonadotropins. These findings implicate SF-1 as a cell-specific transcription factor that may regulate gonadotroph differentiation in the pituitary.

Testosterone is required for gonadotropin-releasing hormone stimulation of luteinizing hormone-beta messenger ribonucleic acid expression in female rats.

Pulsatile GnRH stimulates the synthesis and secretion of LH and FSH in both male and female rats. In the male rat, exogenous GnRH pulses increase alpha, LH and FSH beta messenger RNAs (mRNAs) 3-fold within 24 h. In contrast, the results of recent in vivo and in vitro studies have shown that GnRH stimulates an increase in alpha and FSH beta mRNAs, but not LHbeta. However, during the estrous cycle, LHbeta mRNA increases during the GnRH-induced LH surge on proestrus afternoon. This increase in LHbeta mRNA appears to be coincident with a transient rise in serum testosterone (T). Therefore, the present study was conducted to determine whether T has a role in facilitating GnRH stimulation of LHbeta mRNA expression. In the first group of studies, adult female rats were ovariectomized, and T implants were inserted sc 7 days before the study (serum T, 1.86 ng/ml). Animals received iv pulses of GnRH (25 ng; 30-min interval) for 6-24 h (saline pulses to controls). The data showed that in the presence of T, GnRH stimulated a significant increase in LHbeta (as well as alpha and FSH beta) mRNAs within 6 h (P < 0.05 vs. saline-pulsed controls). Other results revealed that T treatment was critical to the stimulatory effect of GnRH on LH beta mRNA. A second group of studies examined the time course and dose effects of T on LH beta mRNA expression. Maximal LH beta mRNA responses to GnRH (3-fold increase vs. saline controls; P < 0.05) were seen after pretreatment with the lowest dose of T examined (serum T, 0.42 ng/ml), which is similar to T concentrations on proestrus. Higher doses of T suppressed LH release, as well as LH mRNA responses to GnRH. The T-induced LHbeta mRNA response to pulsatile GnRH was seen within 24 h of exposure to T and was the result of an androgenic action, as similar results were observed in rats that received dihydrotestosterone. These findings suggest that T is required to facilitate GnRH stimulation of LHbeta mRNA in the female rat. Moreover, in the presence of the concentrations of T seen on proestrus, LHbeta mRNA increases within 6 h, which is similar to the time course seen during the LH surge. Thus, the present results also suggest that the combined effects of the rise in serum T and increased GnRH secretion induce the rapid rise in LHbeta mRNA expression on the afternoon of proestrus.

Expression of gonadotropin-releasing hormone (GnRH) receptor gene is altered by GnRH agonist desensitization in a manner similar to that of gonadotropin beta-subunit genes in normal and castrated rat pituitary.

It was previously established that the administration of a potent GnRH agonist such as triptorelin (D-Trp6-GnRH) induced desensitization of pituitary gonadotropic cells, resulting in decreased expression of gonadotropin beta-subunit genes and the suppression of LH and FSH synthesis and release. Binding of GnRH to the pituitary is also affected by agonist treatment. To examine the desensitizing effects of GnRH agonist on the expression of the pituitary GnRH receptor (GnRH-R) gene, male rats were given triptorelin (long-acting formulation, 300 micrograms/kg), and levels of GnRH-R messenger RNA (mRNA) were determined by Northern and dot blot hybridization to a 32P-labeled rat complementary DNA probe. Abundances of gonadotropin alpha-subunit, LH beta, and FSH beta mRNAs were examined in parallel, using appropriate probes. A rapid time-dependent decrease in the level of GnRH-R mRNA was observed in rats after triptorelin administration. A minimum residual level of mRNA, in the range of 20-25% of the initial value, was attained as early as 5 h after treatment. Levels further stabilized to 25-30% after a small transient increase to 45% on day 5. A single injection was effective for at least 30 days, after which GnRH-R mRNA levels slowly returned to normal, suggesting a progressive abolition of agonist effects. A concomitant acute depletion of mRNA levels was observed for LH beta and FSH beta (50% decrease in about 48 and 3 h, respectively), whereas the alpha-subunit message increased (rapidly reaching a level 1.8-fold that in control rats after 1-2 days). Castration induced a 3.8-fold elevation in the amounts of GnRH-R mRNA after 3 weeks, whereas alpha, LH beta, and FSH beta mRNAs increased by 6.2-, 7.9-, and 4.2-fold, respectively, compared to corresponding values in intact animals. Administration of the GnRH agonist readily prevented, for as long as 3 weeks, the stimulatory effects of castration on the GnRH-R mRNA and mRNAs for the beta-subunit of gonadotropins, but not for the alpha mRNA, which remained at a high level. When triptorelin was administered 3 weeks postoperatively, the castration-induced increase in LH beta and FSH beta was totally abolished, and no significant effect was noted on alpha-subunit mRNA. In conclusion, these data demonstrate that expression of the GnRH-R gene is subject to regulation and depends on GnRH stimulation, in a manner that indicates susceptibility to desensitizing action by the long-acting GnRH analog, triptorelin.(ABSTRACT TRUNCATED AT 400 WORDS)

Transcriptional activation of the follicle-stimulating hormone beta-subunit gene by activin.

Activin markedly stimulates FSH beta messenger RNA (mRNA) levels in rat pituitary cells. Nonetheless, the molecular mechanisms through which activin enhances FSH beta gene expression are not clear. To assess the role of transcriptional activation in activin stimulation, we first transfected two -2300FSH beta Luc constructs into primary pituitary cells and treated them with activin in plated culture and perifusion. Basal expression of the constructs was low, and no activin response was observed. These results suggested that additional FSH beta sequences are required for basal expression or that the effects of activin are not transcriptional. An alternative approach for measuring transcriptional responses was developed based upon changes in the levels of FSH beta primary transcripts (FSH beta-PT; newly transcribed mRNAs that still contain the first intron) after activin (3 ng/ml) stimulation of perifused rat pituitary cells. An increase in FSH beta-PT was observed after 30 min of activin stimulation, preceding the first observable rise in mature FSH beta mRNA. Levels of FSH beta-PT peaked between 1-2 h, then fell to a lower level (28% of maximal) at 4 h, which was maintained through 10 h of activin stimulation (36% of maximal at 10 h). Mature FSH beta mRNA levels peaked between 2-4 h, which is after the increase in FSH beta-PT, and fell more gradually between 4-10 h of stimulation (56% of maximal at 10 h). Unstimulated levels of mature mRNA and FSH beta-PT did not vary significantly over the course of the experiments. Cotreatment with the transcriptional inhibitor actinomycin-D (2 microM) blocked activin stimulation of both FSH beta-PT and FSH beta mRNA, confirming the transcriptional basis for these events. In summary, we have documented rapid and sequential increases in FSH beta-PT and mature FSH beta mRNAs after activin stimulation, which are prevented by transcriptional blockade. These data provide evidence that the increase in FSH beta mRNA levels after activin treatment are at least partly due to transcriptional activation of the FSH beta gene.

Effects of pulsatile pituitary adenylate cyclase activating polypeptide (PACAP) on gonadotropin secretion and subunit mRNA levels in perifused rat pituitary cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) administered intermittently on gonadotropin secretion and subunit mRNA levels in anterior pituitary cells from 7wk old intact and orchidectomized rats were studied. Cells were perifused for 9 h with hourly pulses of 2.5 nM GnRH or 10 nM PACAP, or with medium alone. Pulsatile PACAP initiated episodes of LH, FSH and alpha-subunit secretion, but was much less effective than GnRH. Responsiveness declined with repeated pulses of PACAP, although interpulse secretion increased gradually throughout the experiment. PACAP was a slightly more effective stimulator of LH release by pituitary cells from castrated than intact rats. At the completion of perifusion, pituitary RNA from castrated rats was extracted for measurement of gonadotropin subunit mRNA levels. Pulsatile PACAP stimulated alpha-subunit and LHbeta mRNA levels but did not affect FSHbeta mRNA. By contrast, continuous PACAP increased alpha-subunit mRNA levels, but suppressed FSHbeta mRNA without affecting LHbeta mRNA. We conclude that pulsatile PACAP is a relatively ineffective stimulator of gonadotropin secretion when administered alone, and regulates gonadotopin subunit gene expression quite differently than continuous PACAP. We propose that the mode of PACAP secretion in vivo may be a determinant of the differential expression of the gonadotropin subunit genes.

Regulation of gonadotropin, thyrotropin subunit, and prolactin messenger ribonucleic acid expression by pulsatile or continuous protein kinase-C stimulation.

The present study investigated the importance of pulsatile vs. continuous protein kinase-C (PKC) stimulation in regulating pituitary gene expression. Adult female rat pituitaries were dissociated, and cells were plated for 48 h, then inserted into perifusion chambers (n = 5-8/group). Chambers received pulses of GnRH (100 pM) plus TRH (4 nM) or sn-1,2-dioctanoylglycerol (DOG; peak chamber concentration, 0.2, 1, or 5 mM; vehicle pulses to controls) every 60 min or a continuous infusion of phorbol 12-myristate 13-acetate (PMA; 20 nM). Secretory responses were determined in perifusate fractions collected after 2 and 22 h of perifusion. After 24 h of treatment, the cells were recovered, total RNA was extracted, and messenger RNAs (mRNAs) were measured by dot blot hybridization. The data revealed that GnRH plus TRH and both pulsatile (DOG) and continuous (PMA) PKC stimulation increased LH, FSH, TSH, and PRL secretory activity. Pulses of GnRH plus TRH increased PRL, alpha, TSH beta, and FSH beta mRNAs, but not LH beta mRNA. Pulsatile DOG only increased LH beta and PRL mRNAs, with maximal responses seen after the 1-mM dose for LH beta and the 0.2-mM dose for PRL. In contrast, PMA stimulated significant increases in alpha, LH beta, and TSH beta, but not PRL or FSH beta. These data show that alpha, TSH beta, LH beta, and PRL mRNA expression are regulated by PKC. Maximal increases are seen after continuous stimulation (via PMA), with the exception of PRL, which requires a pulsatile signal pattern. Thus, intermittent activation of PKC does not appear to play a major role in regulating pituitary gene expression.

Hormonal regulation of human follicle-stimulating hormone-beta subunit gene expression: GnRH stimulation and GnRH-independent androgen inhibition.

Previous studies from our laboratory demonstrated that chronic testosterone administration to castrated transgenic mice suppressed human follicle-stimulating hormone-beta (FSH beta) mRNA levels transcribed from a human transgene to approximately 20% of control values. In the present study we used primary pituitary cultures prepared from the transgenic mice and in vivo experiments in hypogonadal (hpg) mice carrying the human transgene to assess the role of hypothalamic gonadotropin-releasing hormone (GnRH) in this inhibitory action. The levels of human FSH beta mRNA in monolayer cultures of pituitary cells were decreased by 24-hour treatments with 10 nM testosterone propionate or 5 alpha-dihydrotestosterone to 13 and 26% of control values, respectively, in the absence of GnRH. For the in vivo experiments we introduced the 10-kb human FSH beta transgene into the hpg genetic background by selective crossbreeding. Daily injections of 1 microgram GnRH for 14 days induced expression of the human FSH beta gene in male and female mice. Maximal effects were obtained by GnRH treatment of gonadectomized, hpg transgenic mice. Human FSH beta mRNA levels rose to approximately 4- or 10-fold that of control males and females, respectively. The stimulation was blocked completely by simultaneous administration of testosterone propionate in males and partially by estradiol in females. Pituitary content of immunoreactive FSH paralleled the mRNA changes. These data suggest that testosterone feedback inhibits the human FSH beta subunit gene directly at the pituitary gland in addition to the indirect mechanism of GnRH suppression. Furthermore, the in vitro data indicate that the suppression of human FSH beta gene expression is at least partly a direct androgen effect that does not require aromatization of testosterone to estradiol.

Gonadotropin-releasing hormone pulse frequency regulates expression of pituitary follistatin messenger ribonucleic acid: a mechanism for differential gonadotrope function.

Follistatin (FS) is a monomeric glycoprotein that selectively inhibits both secretion of FSH and expression of FSH beta messenger RNA (mRNA), presumably via its ability to bind activin. FS mRNA and protein are present in the gonadotrope, suggesting a local action in regulating FSH beta. Pituitary FS mRNA increases after gonadectomy and at the midcycle gonadotropin surge of the estrous cycle, times of increased GnRH secretion. Thus, the purpose of the present studies was to assess the role of GnRH secretion on the regulation of pituitary FS. To confirm GnRH regulation of FS and to study the role of gonadal steroids, adult male rats were gonadectomized (2-36 h), with some animals receiving either testosterone (T) replacement, LRF-147 (a GnRH antagonist, AC-DTrp1-pCl-DPhe2-DTrp3-Ser4-Tyr5-DArg6-L eu7-Arg8-Pro9-DAla10), or both for 36 h (from the time of castration). Pituitary FS mRNA increased rapidly after castration, with levels rising 3-fold by 12 h and 4-fold by 36 h when compared to intact animals (P < 0.05). This rise was completely abolished by administration of LRF-147 and prevented by T replacement. Because GnRH pulse frequency can selectively regulate FSH beta mRNA expression, we next examined the effect of GnRH pulse interval (8-480 min) on FS mRNA expression. Fast frequency GnRH pulses (8 min), which did not increase FSH beta mRNA, were associated with an increase in FS mRNA (2.5-fold). The 30-min interval increased FS and gonadotropin subunit mRNAs. Slower pulse frequencies (> or = 120 min), which selectively stimulated a rise in FSH beta mRNA, did not increase FS mRNA. These results indicate that pituitary FS mRNA is regulated by GnRH. In addition, GnRH frequency modulation of pituitary FS provides a mechanism whereby a single hypothalamic GnRH can differentially regulate the gonadotropins, LH and FSH.

Effects of pituitary adenylate cyclase-activating polypeptide on gonadotropin secretion and subunit messenger ribonucleic acids in perifused rat pituitary cells.

Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a neuropeptide related to vasoactive intestinal peptide-secretin-glucagon which stimulates adenylate cyclase in cultured rat pituitary cells and stimulates LH and FSH release in vitro and in vivo. Because the cAMP-protein kinase-A pathway regulates the gonadotropin subunit messenger RNAs (mRNAs) and modulates GnRH-stimulated gonadotropin secretion in vitro, we examined the effects of PACAP38 on gonadotropin secretion and subunit mRNA levels. Anterior pituitary cells were prepared from 7-week-old male rats castrated at 5 weeks of age. In monolayer cultures stimulated with GnRH, 0.1-10 nM PACAP38 decreased (P < 0.05) the EC50 for GnRH dose-dependently without affecting the maximum LH secretory response. Cells were next stimulated with 1-min pulses of 2.5 nM GnRH every hour for 9 h in the absence or presence of 10 nM PACAP38, which was perifused continuously. The amplitude of GnRH-induced LH, FSH, and alpha-subunit secretory episodes from PACAP38-treated cells rose (P < 0.01) gradually to 233 +/- 54%, 197 +/- 44%, and 378 +/- 104%, respectively (mean +/- SEM; n = 5 experiments), of the value for control cells lacking PACAP38. This enhancement was sustained for at least 3 h after PACAP38 was removed from the perifusion medium. With PACAP treatment, interpulse secretion of LH and alpha-subunit increased gradually (P < 0.01) to 174 +/- 21% and 212 +/- 64% of the value for chambers stimulated with GnRH alone (control), respectively, whereas interpulse secretion of FSH declined (P < 0.001) to 75 +/- 7% of the control value. In contrast to the gradual effect of PACAP38 to enhance GnRH-induced hormone secretion, PACAP38 alone produced a transient burst of gonadotropin secretion. At the completion of the perifusions, total RNA was extracted and gonadotropin subunit mRNA levels were determined by Northern analysis. GnRH increased (P < 0.01) FSH beta mRNA to 438 +/- 52% of the level in cells stimulated with medium alone (control). Adding PACAP38 to the perifusion medium partially blocked (P < 0.01) the effect of GnRH (178 +/- 20% of the control value), and PACAP38 alone reduced (P < 0.01) FSH beta mRNA levels to 31 +/- 3% of the control value. By contrast, alpha-subunit mRNA levels were increased by both PACAP38 (143 +/- 4% of the control value; P < 0.01) and GnRH (121 +/- 2% of the control value; P < 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Ovarian regulation of pituitary inhibin subunit and activin receptor type II gene expression: evidence for a nonsteroidal inhibitory substance

Gonadotropin subunit gene expression is regulated by gonadal, hypothalamic, and locally derived hormones. In particular, activin rapidly (within hours) acts at the gonadotrope to selectively increase the expression of FSH beta messenger RNA (mRNA). A family of activin receptors (ActRI, ActRII, and ActRIIB) has been identified, which is expressed in the pituitary as well as numerous other tissues in which activin is thought to act. As alterations in activin sensitivity could modulate activin action and, thereby, FSH beta mRNA, the purpose of this study was to determine whether ovariectomy (OVX), which results in rapid (< 2 h) increases in FSH beta, is associated with changes in ActRII gene expression. Adult female rats were ovariectomized, and some animals also received a GnRH antagonist from the time of OVX. Animals were killed between 2 h and 7 days later, and ActRII mRNA levels were measured by a quantitative reverse transcriptase-polymerase chain reaction assay. Although levels were unchanged at 2 h, ActRII mRNA levels increased 5- to 6-fold by 8 h and remained increased through 7 days after OVX. These changes were not altered by GnRH blockade. To determine whether ActRII was regulated by gonadal steroids, female rats were ovariectomized, and some animals were replaced with estradiol and progesterone (Silastic implants) for 2 days. Again, ActRII mRNA levels increased significantly after OVX, and gonadal steroid replacement had no effect. Finally, to investigate whether pituitary ActRII mRNAs are regulated by circulating inhibin, intact female rats were treated with an inhibin antiserum or nonimmune sheep serum as a control and killed 12 h later. Despite its action to increase FSH beta mRNA and FSH secretion, selective removal of inhibin did not alter ActRII mRNA levels. Based on these results we conclude the following. 1) Pituitary ActRII mRNAs increase rapidly after OVX, although increases in FSH beta precede changes in ActRII. These data suggest that changes in activin sensitivity may be a factor involved in the regulation of FSH beta. 2) An ovarian factor, other than inhibin, estradiol, and progesterone, acting independently of GnRH maintains an inhibitory tone on pituitary ActRII gene expression in adult rats.

Ovarian regulation of pituitary inhibin subunit and activin receptor type II gene expression: evidence for a nonsteroidal inhibitory substance.

Gonadotropin subunit gene expression is regulated by gonadal, hypothalamic, and locally derived hormones. In particular, activin rapidly (within hours) acts at the gonadotrope to selectively increase the expression of FSH beta messenger RNA (mRNA). A family of activin receptors (ActRI, ActRII, and ActRIIB) has been identified, which is expressed in the pituitary as well as numerous other tissues in which activin is thought to act. As alterations in activin sensitivity could modulate activin action and, thereby, FSH beta mRNA, the purpose of this study was to determine whether ovariectomy (OVX), which results in rapid (< 2 h) increases in FSH beta, is associated with changes in ActRII gene expression. Adult female rats were ovariectomized, and some animals also received a GnRH antagonist from the time of OVX. Animals were killed between 2 h and 7 days later, and ActRII mRNA levels were measured by a quantitative reverse transcriptase-polymerase chain reaction assay. Although levels were unchanged at 2 h, ActRII mRNA levels increased 5- to 6-fold by 8 h and remained increased through 7 days after OVX. These changes were not altered by GnRH blockade. To determine whether ActRII was regulated by gonadal steroids, female rats were ovariectomized, and some animals were replaced with estradiol and progesterone (Silastic implants) for 2 days. Again, ActRII mRNA levels increased significantly after OVX, and gonadal steroid replacement had no effect. Finally, to investigate whether pituitary ActRII mRNAs are regulated by circulating inhibin, intact female rats were treated with an inhibin antiserum or nonimmune sheep serum as a control and killed 12 h later. Despite its action to increase FSH beta mRNA and FSH secretion, selective removal of inhibin did not alter ActRII mRNA levels. Based on these results we conclude the following. 1) Pituitary ActRII mRNAs increase rapidly after OVX, although increases in FSH beta precede changes in ActRII. These data suggest that changes in activin sensitivity may be a factor involved in the regulation of FSH beta. 2) An ovarian factor, other than inhibin, estradiol, and progesterone, acting independently of GnRH maintains an inhibitory tone on pituitary ActRII gene expression in adult rats.

Cytochemical detection of gonadotropin-releasing hormone-binding sites on rat pituitary cells with luteinizing hormone, follicle-stimulating hormone, and growth hormone antigens during diestrous up-regulation.

Pituitary cells with GnRH receptors increase over 2-fold during diestrus to reach a peak during the morning of proestrus. This is followed by a rapid fall during the afternoon of proestrus to reach a nadir by estrus. The objective of this study was to learn the identity of the new target cells added during diestrus. This was particularly important in view of recent evidence showing that gonadotropes with LH beta and FSH beta mRNA have GH antigens. Pituitary cells from diestrous and proestrous rats were exposed to biotinylated GnRH (Bio-GnRH) for 10 min. Bio-GnRH was detected by avidin peroxidase, and then the cells were immunolabeled for pituitary hormones. The percentages of cells labeled for Bio-GnRH rose during diestrus from 6.6 +/- 0.8% in the morning to 11.9 +/- 0.7% by evening (mean +/- SD). By the morning of proestrus, the percentages of Bio-GnRH target cells increased further to 16 +/- 0.7%. The percentages of pituitary cells dual labeled for LH beta antigens and Bio-GnRH rose from 4.3 +/- 0.6% to 9% +/- 1% during diestrus and averaged 13 +/- 0.7% by the morning of proestrus. At this time, 90% of cells with LH antigens bound Bio-GnRH. When percentages of pituitary cells with FSH beta antigens and Bio-GnRH-binding sites were analyzed, there was an increase during diestrus from 4 +/- 0.4% to 9.7 +/- 0.7%; a peak level of 14 +/- 0.9% was reached by the morning of proestrus. Bio-GnRH binding was expressed by 86% of FSH cells during this peak. Finally, GH antigens were also detected in GnRH target cells. The percentage of cells dual labeled for Bio-GnRH and GH increased from 4 +/- 0.8% to 8 +/- 1% during diestrus and the morning of proestrus. During the diestrous and proestrous peak periods of expression, Bio-GnRH binding was seen in 32% of GH cells. None of the other pituitary cell types showed significant GnRH binding. These studies showed that most of the new GnRH-receptive cells stem from maturing gonadotropes. Half of the GnRH-receptive cells also contain GH antigens, which correlated with results from previous studies that showed GH antigens in cells with gonadotropin mRNAs. This might reflect expression of gonadotrope functions by a subset of GH cells. Alternatively, the GH antigens may be bound to GH receptors in gonadotropes. This latter possibility may signify a paracrine regulation of gonadotrope function by GH.

Cytochemical detection of gonadotropin-releasing hormone-binding sites on rat pituitary cells with luteinizing hormone, follicle-stimulating hormone, and growth hormone antigens during diestrous up-regulation

Pituitary cells with GnRH receptors increase over 2-fold during diestrus to reach a peak during the morning of proestrus. This is followed by a rapid fall during the afternoon of proestrus to reach a nadir by estrus. The objective of this study was to learn the identity of the new target cells added during diestrus. This was particularly important in view of recent evidence showing that gonadotropes with LH beta and FSH beta mRNA have GH antigens. Pituitary cells from diestrous and proestrous rats were exposed to biotinylated GnRH (Bio-GnRH) for 10 min. Bio-GnRH was detected by avidin peroxidase, and then the cells were immunolabeled for pituitary hormones. The percentages of cells labeled for Bio-GnRH rose during diestrus from 6.6 +/- 0.8% in the morning to 11.9 +/- 0.7% by evening (mean +/- SD). By the morning of proestrus, the percentages of Bio-GnRH target cells increased further to 16 +/- 0.7%. The percentages of pituitary cells dual labeled for LH beta antigens and Bio-GnRH rose from 4.3 +/- 0.6% to 9% +/- 1% during diestrus and averaged 13 +/- 0.7% by the morning of proestrus. At this time, 90% of cells with LH antigens bound Bio-GnRH. When percentages of pituitary cells with FSH beta antigens and Bio-GnRH-binding sites were analyzed, there was an increase during diestrus from 4 +/- 0.4% to 9.7 +/- 0.7%; a peak level of 14 +/- 0.9% was reached by the morning of proestrus. Bio-GnRH binding was expressed by 86% of FSH cells during this peak. Finally, GH antigens were also detected in GnRH target cells. The percentage of cells dual labeled for Bio-GnRH and GH increased from 4 +/- 0.8% to 8 +/- 1% during diestrus and the morning of proestrus. During the diestrous and proestrous peak periods of expression, Bio-GnRH binding was seen in 32% of GH cells. None of the other pituitary cell types showed significant GnRH binding. These studies showed that most of the new GnRH-receptive cells stem from maturing gonadotropes. Half of the GnRH-receptive cells also contain GH antigens, which correlated with results from previous studies that showed GH antigens in cells with gonadotropin mRNAs. This might reflect expression of gonadotrope functions by a subset of GH cells. Alternatively, the GH antigens may be bound to GH receptors in gonadotropes. This latter possibility may signify a paracrine regulation of gonadotrope function by GH.

Regulation of gonadotropin mRNA levels in cultured rat pituitary cells by gonadotropin-releasing hormone (GnRH): role for Ca2+ and protein kinase C.

Incubation of cultured rat pituitary cells with gonadotropin-releasing hormone (GnRH, 1 nM) resulted in a rapid elevation of gonadotropin subunit steady-state mRNA levels(alpha, 2.2-fold, LH beta, 2.1-fold, and FSH beta 2.2-fold increases at 30 min). Addition of actinomycin D abolished the stimulatory effect of GnRH upon alpha and LH beta and reduced the effect upon FSH beta mRNA levels. The effect of GnRH is biphasic, where the early phase is being observed at 30-60 min, while the late phase is noticed between 12-24 h. A significant decrease in FSH beta mRNA levels was found after 6 h of incubation when using a stable GnRH analog. The unique profile of the time response enabled us to attempt to dissect the signal transduction cascade involved in the neurohormone action. Addition of the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the Ca2+ ionophore, ionomycin, mimicked the profile of GnRH-induced alpha and LH beta mRNA elevation. The two phases of FSH beta mRNA elevation induced by GnRH could be mimicked by TPA, while the decrease at 6 h was mimicked by ionomycin. The rapid stimulatory effect of GnRH on gonadotropin subunit mRNA levels was abolished by the PKC inhibitors, staurosporine and GF 109203X. Similarly, the rapid stimulatory effect of GnRH on alpha and LH beta, but not FSH beta, was abolished in Ca(2+)-free medium. While additivity in LH release is obtained upon the combined addition of TPA and ionomycin for 30 min of incubation, LH beta and FSH beta gene expression is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle.

FSH synthesis and secretion are regulated by a complex interplay of hypothalamic, gonadal, and pituitary factors. Recent evidence suggests that inhibin, activin, and follistatin, although originally identified as gonadal peptides, are also expressed in the pituitary, where they may be secreted and play an autocrine/paracrine role in the control of FSH beta gene expression. Attempts to study pituitary regulation of the genes encoding these proteins have been hampered by low levels of mRNA expression. Consequently, we developed quantitative reverse transcription-polymerase chain reaction assays for follistatin (FS) and the three subunits (alpha, beta A, and beta B) that comprise activin and inhibin. Expression of activin type II receptor (actRII) mRNA was also analyzed. Reasoning that mRNA levels of these FSH-regulating pituitary peptides might be modulated at times of increased FSH gene expression, two in vivo models were chosen for further investigation. 1) Two weeks after ovariectomy, rat pituitary FS mRNA levels increased substantially (4.09-fold vs. intact) with a modest increase in beta B-inhibin mRNA levels (1.88-fold vs. intact). No changes in alpha-inhibin, beta A-inhibin, or actRII mRNA levels were observed. 2) During the estrous cycle, pituitary FS gene expression varied strikingly, with a peak at 1800 h on proestrus (13.69-fold vs. 0900 h on proestrus), followed by a rapid decline at 2400 h on proestrus (2.10-fold vs. 0900 h on proestrus). A more detailed analysis of expression during proestrus revealed that peak FS mRNA levels preceded peak FSH beta gene expression by 6 h. Levels of the inhibin subunit and actRII transcripts varied minimally across the estrous cycle. We conclude that during the estrous cycle in rats, pituitary FS mRNA levels are regulated dynamically, whereas levels of inhibin/activin subunits and the activin receptor, actRII, very minimally. The observation that FS mRNA levels peak before maximal expression of FSH beta mRNA raises the possibility that FS facilitates, rather than inhibits, FSH biosynthesis in vivo.

Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle

FSH synthesis and secretion are regulated by a complex interplay of hypothalamic, gonadal, and pituitary factors. Recent evidence suggests that inhibin, activin, and follistatin, although originally identified as gonadal peptides, are also expressed in the pituitary, where they may be secreted and play an autocrine/paracrine role in the control of FSH beta gene expression. Attempts to study pituitary regulation of the genes encoding these proteins have been hampered by low levels of mRNA expression. Consequently, we developed quantitative reverse transcription-polymerase chain reaction assays for follistatin (FS) and the three subunits (alpha, beta A, and beta B) that comprise activin and inhibin. Expression of activin type II receptor (actRII) mRNA was also analyzed. Reasoning that mRNA levels of these FSH-regulating pituitary peptides might be modulated at times of increased FSH gene expression, two in vivo models were chosen for further investigation. 1) Two weeks after ovariectomy, rat pituitary FS mRNA levels increased substantially (4.09-fold vs. intact) with a modest increase in beta B-inhibin mRNA levels (1.88-fold vs. intact). No changes in alpha-inhibin, beta A-inhibin, or actRII mRNA levels were observed. 2) During the estrous cycle, pituitary FS gene expression varied strikingly, with a peak at 1800 h on proestrus (13.69-fold vs. 0900 h on proestrus), followed by a rapid decline at 2400 h on proestrus (2.10-fold vs. 0900 h on proestrus). A more detailed analysis of expression during proestrus revealed that peak FS mRNA levels preceded peak FSH beta gene expression by 6 h. Levels of the inhibin subunit and actRII transcripts varied minimally across the estrous cycle. We conclude that during the estrous cycle in rats, pituitary FS mRNA levels are regulated dynamically, whereas levels of inhibin/activin subunits and the activin receptor, actRII, very minimally. The observation that FS mRNA levels peak before maximal expression of FSH beta mRNA raises the possibility that FS facilitates, rather than inhibits, FSH biosynthesis in vivo.

Role of growth factors and estrogen as modulators of growth, differentiation, and expression of gonadotropin subunit genes in primary cultured sheep pituitary cells.

Although the pituitary is known to produce several growth factors, their effects on pituitary cell growth and differentiation are still unclear, particularly in normal tissue. Using primary cultures of aged ewe pituitaries cultured in serum-free conditions, we studied the effects of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-beta (TGF beta 1), insulin, and 17 beta-estradiol (E2) on the growth, differentiation, and expression of gonadotropin subunit genes. After 72-h incubation of the monolayer (day 5) with optimal concentrations of each factor, [3H]thymidine incorporation was increased significantly (P < 0.01) over the control values by 33 +/- 8% (mean +/- SEM; n = 3; 10 nM E2), 36 +/- 10% (1 ng/ml TGF beta 1), 83 +/- 12% (10 ng/ml bFGF), and 118 +/- 12% (1 nM EGF). Insulin showed a two-phase dose-response curve, increasing [3H]thymidine uptake by 34 +/- 9% at 10 ng/ml and by 63 +/- 13% at 10 micrograms/ml. Cell counting using a Coulter counter confirmed these results. Characterization of cell types by immunocytochemistry (avidin-biotin-peroxidase complex technique) revealed that the cell cultures were predominantly gonadotrophs. However, the cultures contained cells that did not stain with any specific ovine or human antiserum against LH beta, FSH, TSH beta, PRL, GH, ACTH, or glial fibrillary acidic protein, but were of epithelial cell lineage, as shown by positive keratin staining. Treatment with EGF and bFGF increased the proportion of these undifferentiated pituitary cells and induced changes in their morphology to large cuboidal cells containing large nuclei. After treatment with E2, insulin, and TGF beta 1, pituitary cells remained differentiated, although with E2, staining for gonadotrophs was much reduced. Northern blot analysis revealed that E2 treatment for 0-48 h progressively reduced the mRNA for FSH beta (16 +/- 4.5% of control values) and LH beta (12.4 +/- 2.5%), but had little effect on the common alpha-subunit (88.4 +/- 4.6%). TGF beta 1, however, stimulated the expression of FSH beta subunit gene by 142 +/- 4.6% (P < 0.01) of the control value, but had no significant effect on LH beta and common alpha-subunit genes. Insulin, EGF, and bFGF showed no significant effect on the expression of these three subunit genes. The data define the direct effects of growth factors and E2 on the growth and differentiation of normal sheep pituitary cells and gonadotrophs in particular, which may be of relevance to the pathophysiology of the pituitary and in the multistage process of pituitary tumorigenesis.

Testosterone feedback on gonadotropin secretion and gene expression in transgenic mice expressing human growth hormone gene.

To determine the effects of testosterone on the regulation of gonadotropins in metallothionein-1/human growth hormone (MT/hGH) transgenic mice, basal and gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from incubated pituitaries, as well as pituitary content of LH, FSH, and mRNA for their respective beta subunits, were measured in normal and transgenic males that were injected with testosterone propionate (5 micrograms/g body weight; 24 hours before autopsy), injected with oil vehicle, castrated for 10 days, or sham operated. In normal (non-transgenic) males, exogenous testosterone induced the expected suppression, and castration induced the expected stimulation of various parameters of gonadotropin synthesis and release. In contrast, in testosterone-treated and in castrated MT/hGH transgenic mice the release of LH and the pituitary levels of LH-beta mRNA did not differ from the corresponding values measured in vehicle-injected and sham-operated transgenic controls. Pituitary LH content was elevated in testosterone-treated MT/hGH transgenic mice but was not changed in castrated transgenic males. The changes in pituitary levels of FSH and FSH-beta mRNA and in FSH release in MT/hGH transgenic mice in response to testosterone and castration were different from the changes in LH and LH-beta mRNA in the same mice, but similar to the changes of FSH and FSH-beta message produced in normal mice by identical treatments. We suggest that hGH expression attenuates the effects of testosterone on the mechanisms controlling LH release, with less influence on testosterone regulation of LH synthesis. These effects of hGH expression appear to be selective for LH, without influencing the FSH control system.

The effect of in vitro ethanol exposure on luteinizing hormone and follicle stimulating hormone mRNA levels, content, and secretion.

It has been previously shown that acute ethanol (EtOH) exposure in vivo resulted in suppression of serum LH and pituitary beta-LH subunit mRNA levels in castrated male rats. While serum FSH levels also were noted to fall after in vivo, the mRNA for beta-FSH was not altered. The aim of the present studies was to determine whether these effects could be accounted for by a direct EtOH effect at pituitary level. To this end we examined the direct effect of EtOH on LH and FSH synthesis and secretion utilizing dispersed anterior pituitary cells from gonadectomized adult male rats. After a 72 hour post dissociation healing period, the cells were exposed to media containing 0 or 200 mg% EtOH for one hour. The media was removed and the cells incubated with EtOH-free media for an additional 1, 3 or 6 hrs. In the EtOH exposed cells, secretion of both LH and FSH increased to > 300% (p < 0.001) of control. At 6 hrs after withdrawal of EtOH a significant reduction in both LH and FSH secretion was seen. Intracellular content of LH and FSH was unchanged before and after withdrawal of EtOH. Steady state levels of beta LH and beta FSH mRNA were unchanged at all time points. In a separate series of experiments, pituitary cells from gonadectomized adult male rats were continuously exposed to different concentrations of EtOH ranging from 0-400 mg% for 3 hrs. LH secretion was stimulated by 400 mgm% EtOH only, while the intracellular content of LH was significantly reduced with the 400 mg% dose. The secretion of FSH was stimulated by 200 mg% and 400 mg% high dose EtOH after 3 hours, with concomitant reduction in FSH pituitary content at both these EtOh dose levels. The mRNA for both beta-LH and beta FSH was not different with any dose of EtOH compared to levels of control, non-EtOH exposed cells. We conclude that though there were similarities between in vivo and in vitro LH and FSH responses to EtOH, the differences reported here indicate that the in vivo responses are not totally explained by a direct EtOH effect at pituitary level. Rather, they must, in addition, reflect action at suprapituitary site(s), pituitary effects of EtOH metabolites or condensation products, and/or alterations in LH and FSH clearance.